The MAGT1 transporter is critically involved in the selective regulation of intracellular free Mg2+ levels in mammalian cells. The molecular functions of free Mg2+ in eukaryotic cells have not been fully established. We found that patients with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. In studying lymphocytes from these patients, we found that a deficiency of MAGT1 caused decreased basal intracellular free Mg2+ leading to defective expression of the natural killer activating receptor NKG2D in NK and CD8+ T cells. Without NKG2D, cytolytic responses against EBV are diminished, thereby revealing the first specific molecular function of intracellular basal free Mg2+ in eukaryotic cells. Moreover, intracellular free Mg2+, NKG2D expression and function can be rescued in vitro by incubating patient cells and elevated levels of Mg2+. Moreover, NKG2D expression and cytolytic function can be improved and EBV-infected cells reduced in vivo, in MAGT1-deficient patients by magnesium administration. Thus, our data indicate an important molecular function for free basal Mg2+ in immunity and demonstrate a requirement for NKG2D cytolytic function in an essential EBV antiviral response in humans. Recent advances in understanding the role of divalent cations in immune cells have uncovered new signaling functions for Mg2+ and Zn2+ opening new vistas for future investigation. Many questions about Mg2+ signaling are still unanswered. In fact, there are a plethora of apparent Mg2+ transport and channel proteins whose role in human physiology is not yet been determined in the healthy and diseased immune system. Mg2+ mobilization has been observed in variety of cell types, however the molecular mechanisms regulating these cation changes remain elusive. Technical limitations such as probe sensitivity, low electrogenicity, and other considerations prevent the precise study of the regulated mobilization of these cations. Our current collaborations with academic and industrial partners are directed at addressing these issues. Another aspect that require further investigation is the understanding of the various effectors of divalent cations signaling and the network(s) that they generate. Beyond enhancing the basic scientific knowledge, answering these questions have also important medical implications especially, but not only, for immune disorders associated with divalent cations signaling deficiency, such as XMEN disease, SOCE deficiency or even dietary Zn2+ deficiency. Thirty years ago, the discovery of the immunosuppressant abilities of the Ca2+ signaling inhibitors, cyclosporine A (CN inhibitor), revolutionized organ transplantation. Thus, uncovering new Mg2+ and Zn2+ sensitive targets or functions could identify new therapeutic targets for immunomodulation. Moreover, the promising results of Mg2+ and Zn2+ supplementation in various pathological settings should encourage the development of new ion supplementation strategies. Also, there are at least 22 known magnesium transporters or channels of which we have detected at least 16 on T lymphocytes. There is very little known about these transporters or channels so this represents an opportunity to understand new fundamental aspects of ion regulation. Since other ion channels have been good pharmaceutical targets, this may open up new areas for therapeutic development. We are especially interested in pursuing additional questions related to the role of Mg2+ in the control of EBV. Despite being linked to both epithelial (nasopharyngeal and gastric) and lymphoid (Burkitt and Hodgkin lymphoma) malignancies, there are currently no known methods for primary or secondary prevention of chronic EBV infection or the associated malignancies. Our discovery that a genetic deficiency of a Mg2+ ion transporter caused a selective immunodeficiency that led to uncontrolled EBV infection and an extremely high rate of EBV+ lymphoma in affected children and that dietary supplementation with Mg2+ (a widely available and inexpensive nutraceutical) could correct the immune defect by increasing a specific antiviral receptor called NKG2D which markedly decreased or eliminated EBV offered a new hypothesis about chronic EBV in Africa. We have begun a collaboration with Sam Mbulaiteye, a National Cancer Institute investigator, who studies EBV/lymphoma risk in Africa. We have carried out a preliminary study of previously collected case-control blood samples from Africa and showed that there was a statistically significant deficiency of serum Mg2+ in Burkitt lymphoma (BL) patients with high EBV. This preliminary study could not answer whether intracellular Mg2+ and NKG2D expression were deficient in these patients which requires flow cytometric analysis on site in Africa or whether these could be restored by adding more Mg2+ to the cells. However, these results held promise that endemic EBV and the consequent lymphomas could be prevented by simple dietary supplementation with Mg2+. The current project aims to investigate whether intracellular Mg2+ and NKG2D expression underlie EBV chronic infection and/or malignancy to develop the rationale to institute dietary supplementation with Mg2+. Specifically, to measure and correlate basal intracellular Mg2+ and NKG2D levels with EBV genome load in blood in African children at risk of BL and in children with BL. This work will be done in the EMBLEM Study at St. Marys Hospital, Lacor in Uganda. We anticipate recruiting 100 consecutive children with BL diagnosed at St. Marys Hospital Lacor over a 12-month period. In addition, we anticipate recruiting about 40 healthy family members (about 200 unique individuals, based on an average household size of 5) from one village where BL incidence is high to investigate the link between basal intracellular magnesium level and EBV viral genome load in blood in apparently healthy people. The data will be collected using standard flow cytometry assays established in the Lenardo lab that will be performed on-site in Uganda. Further samples will be sent to NIH to verify EBV loads and to determine if T cells from the patients in tissue culture show restoration of basal intracellular Mg2+ level and NKG2D receptor levels by flow cytometry after in vitro treatment with Mg2+ supplementation.